1. Technical Field
The present invention relates to a novel L-amino acid oxidase, a method for measuring L-lysine using this novel L-amino acid oxidase, a kit employed in this method, and an enzyme sensor. More particularly, the present invention relates to a variant L-amino acid oxidase with high substrate specificity for L-lysine, a method for measuring L-lysine employing this variant enzyme, an L-lysine measurement kit, and an enzyme sensor.
2. Brief Description of the Related Art
L-lysine, one of the constituent amino acids of proteins, is an essential amino acid that cannot be produced within the body. The concentration of amino acids including L-lysine within the body is kept at homeostasis. However, congenital metabolic anomalies and internal disorders can cause great fluctuation in blood concentrations. The concentration of not just L-lysine, but of other amino acids within the body, affords a useful means of detecting disease. Thus, by measuring the blood concentration of one or many amino acids, it is possible to detect disease (International publication No. WO2006/129513 and Anal. Chem. 81: 307-314 (2009)).
In recent years, a large number of methods employing enzymes have become known as methods of quantifying amino acid levels. These methods employing enzymes are advantageous since they are less expensive and easier to implement than instrumental analysis methods. For example, enzymes such as dehydrogenase and oxidase are often employed. An example of a quantification method employing oxidase is the use of peroxidase to detect the hydrogen peroxide that is produced by subjecting amino acids to the action of oxidase (Japanese Un-examined patent publication No. Shou55-43409). This detection and quantification can be conducted with a method such as the colorimetric method, fluorescence method, or electrode method.
Methods employing enzymes are also known as methods of quantifying L-lysine. For example, in quantification with oxidase, L-lysine α-oxidase [EC 1.4.3.14] has been employed. L-lysine α-oxidase derived from Trichoderma viride affords a higher substrate specificity than other L-amino acid oxidases, and is commercially available. Thus, it has come to be employed in elements such as enzymatic sensors (Guerrieri et al., Sens. Actuators, B 126: 424-430 (2007); Endo et al. Anal. Bioanal. Chem. 391: 1255-1261 (2008); Anal. Bioanal. Chem. 406: 19-23 (2010)).
There are also reports that L-lysine monooxidase derived from Pseudomonas fluorescens exhibits an L-lysine oxygenase activity (Flashner et al., J. Biol. Chem. 249: 2579-2586 (1974); Flashner et al., J. Biol. Chem. 249: 2587-2592 (1974)). This enzyme employs L-lysine, L-threonine, and L-arginine as substrates.
However, the Trichoderma viride-derived L-lysine α-oxidase also exhibits oxidase activity on amino acids other than L-lysine. Thus, when employing L-lysine α-oxidase to quantify a sample containing multiple amino acids, such as blood plasma, excess evaluation tends to be involved.
Furthermore, it has recently been reported that L-lysine α-oxidase derived from the mucus of saltwater fish has greater substrate specificity than the above L-lysine α-oxidase (Endo et al., Anal. Bioanal. Chem. 391: 1255-1261 (2008)). However, this L-lysine α-oxidase is derived from the mucus of saltwater fish, and there are no reports of enzyme production by culturing. Accordingly, it is difficult to produce large quantities of this enzyme for use in quantifying L-lysine.
There have been no reports of using the Pseudomonas fluorescens-derived L-lysine monooxygenase to quantify L-lysine. Even when this enzyme is employed to quantify L-lysine, as set forth above, because it has oxygenase activity on L-ornithine and L-arginine in addition to L-lysine, it is impossible to strictly quantify L-lysine in samples containing multiple amino acids, such as blood plasma.
Methods of quantifying L-lysine based on oxidase, if successful, would be useful from the perspective of being less expensive and more convenient than instrumental analysis methods. However, as stated above, for samples containing multiple amino acids, such as blood plasma, the substrate specificity is low or there are problems with enzyme productivity with respect to the enzymes that are currently available. From this perspective, these enzymes are not practical.